(1) Field of Invention
Subject invention is related to oligomeric mixtures which are active in the protection of oxidative phosphorlyation in degraded mitochondria and exhibit ionophoretic activity. More specifically, this invention relates to the preparation of structurally defined oligomers exhibiting the above activity. It also pertains to structurally modified 15-dehydro-PGB.sub.1 compounds useful as precursors for the said oligomeric mixture. This pertains more specifically to the preparation of the structurally modified 15-dehydro-PGB.sub.1 ; i.e. a "C-16 blocked" 15-dehydro-PGB.sub.1. This invention also pertains to the reaction conditions required for the conversion of said precursor into oligomeric mixtures from which structurally defined dimers and trimers can be isolated.
(2) Statement of Prior Art
The term PGB.sub.x (i.e., an unknown "x" derived from a prostaglandin B, "PGB") has been rather loosely applied to the reaction mixtures derived from extensive base (hydroxide) treatment of a variety of prostaglandin B precursors as described by Polis et al in Physiol. Chem. Physics, 12, 167 (1980) which is incorporated herein by reference. On some occasions, the term PGBx refers to the crude reaction product while at other times it refers to the most active fraction (Fraction 2) derived from size exclusion chromatography of the crude reaction product. Over the last few years, the term PGBx has come to denote a complex mixture derived from treatment of 15-dehydro-prostaglandin B.sub.1 (15-dehydro-PGB.sub.1) methyl ester, with 1N ethanolic potassium hydroxide. Hereinafter, the term "PGBx" will be referred to the most active fraction derived from the size exclusion chromatography of the crude reaction mixture that results from the treatment of 15-dehydro-PGB.sub.1 methyl ester, with 1N ethanolic potassium hydroxide for four hours at 80.degree. C. as will be presently shown in FIG. 1. As will be seen presently in FIG. 2, fraction 2 represents the component exhibiting the maximum biological activity in the protection of oxidative phosphorylation.
A number of unique in vitro and in vivo activities have been demonstrated for certain fractions; i.e., standard PGBx, of the complex mixture derived by ethanolic potassium hydroxide treatment of 15-dehydro-PGB.sub.1, as described by Polis et al and Devlin. Standard PGBx protects against the loss of phosphorylating activity during aging in vitro of rat liver mitochondria and functions as a potent "water soluble" ionophore [as indicated by S. T. Ohnishi and T. M. Devlin, Calcium ionophore activity of a prostaglandin B.sub.1 derivative (PGBx), Biochemical and Biophysical Research Communications, 89, 240 (1979); C. N. Serhan, H. M. Korchak and G. Weissmann; PGBx, a prostaglandin derivative, mimics the action of the calcium ionophore A23187 on human neutrophils, J. Immunol., 80, 2020, (1980); and H. W. Shmukler, Cation complex formation with PGBx, a prostaglandin oligomer, after myocardial infarction with ventricular fibrilation. Effects of PGBx, Physiol. Chem. Physics, 12, 81 (1980)] which stimulates the release of Ca.sup.++ from sacroplasmic reticulum and heart mitochondria. The in vitro activity in the protection of oxidative phosphorylation and ionophoretic activity as demonstrated by Polis et al and Devlin are regarded as predictive of the level of in vivo activity as described below.
In vivo, standard PGBx facilitates and significantly increases survival after what otherwise would be lethal episodes of myocardial ischemia in monkeys and restores nervous system function in dogs after otherwise fatal hypoxia as shown by G. Moss, T. Maglioccheti and R. Quarmby: Immediate restoration of central nervous system autonomic cardiopulmonary control: Survival of "lethal" cerebral hypoxia by treatment with prostaglandin Bx, Surg. Forum, 29, 513 (1978) and R. J. Kolata and B. D. Polis, Facilitation of recovery from ischemic brain damage in rabbits by polymeric prostaglandin PGBx, a mitochondrial protective agent, Physiol. Chem. Physics, 13, 545 (1981). Standard PGBx also provides a significant measure of protection against the severest forms of cardiac ischemia in various isolated segments of canine heart [as shown by A. P. Walls, N. Himori and A. M. Burkman, Comparison of the effects of prostaglandin Bx (PGBx) and verapamil on changes in myocardial function that occur during schemia, Proc. Fed. Am. Soc. Exp. Biol., Apr. 12, 1980, Atlanta, Ga.] and protects isolated anoxic rat heart as shown by S. T. Ohnishi and T. M. Devlin, Protection by PGBx of isolated anoxic rat heart, in their unpublished results. PGBx appears to represent a class of structures which possess a unique ability to prevent or restore damage on a cellular level due to oxygen deprivation although the mechanism of this action remains unclear. Such unique properties strongly suggest potential future application in treatment of incidents of cerebral and myocardial ischemia and as a therapeutic agent for hemmorragic traumatized combat casualties.
Relatively little from a chemical viewpoint had been conclusively established concerning the structural details of PGBx or the chemistry involved in the formation of the active sites. Earlier descriptions of PGBx as a stable free-radical prostaglandin polymer [as suggested by B. D. Polis, E. Polis and S. Kwong: Protection and reactivation of oxidative phosphorylation in mitochondria by a stable free-radical prostaglandin polymer (PGBx), Proc. Natl. Acad. Sci. USA, 76, 1598 (1979)] or even as a polymer derivative of prostaglandin B have been demonstrated to be incorrect upon closer inspection [e.g., G. L. Nelson and G. L. Verdine, The base promoted oligomerization of a 15-dehydro-PGB.sub.1. analog: Structural insights into the complex oligomeric mixture termed PGBx, Tetrahedron Letters, 23, 1967 (1982) and G. L. Nelson and G. L. Verdine, The base promoted oligomerization of 15-dehydro-prostaglandin B1: Dimer formation and structural implications for a complex mixture termed PGBx. Tetrahedron Letters 23, 991 (1983)]. PGBx has been generally characterized on the basis of spectral data as a complex mixture of closely related oligomers formed by an initial reaction at the 13,14-unsaturation of 15-dehydro-PGB.sub.1, with the retention of the overall prostaglandin skeleton as described by Polis et al in the reference cited above. Recent attempts by a number of groups to resolve this complex oligomeric mixture into individual components retaining activity have proven unsuccessful precluding a more definitive structural assignment. For example, synthesis and characterization of polymeric derivatives designated PGBx are disclosed in U.S. Pat. No. 4,153,808 issued May 8, 1979 to David Polis et al; synthesis of prostaglandin analogs including ethyl analog; 3-(trans-3-keto-1-pentenyl)-2-cyclopentenone; is disclosed in U.S. Pat. No. 4,338,466 issued July 6, 1982 to George L. Nelson; methods of preparing prostaglandin B.sub.1 derivatives are discussed in U.S. Pat. No. 4,245,111 issued Jan. 13, 1981 to Polis et al and conversion of prostaglandin analogs into bicarbonate soluble and bicarbonate insoluble oligomeric mixtures are disclosed in my co-pending patent applications Ser. No. 492,087 U.S. Pat. No. 4,668,828 and 492,088 U.S. Pat. No. 4,663,486 and each with a filing date of May 6, 1983. These two patent applications issued as U.S. Pat. Nos. 4,668,828 and 4,663,486, respectively. All these references are incorporated herein by reference. The lack of any definitive structural detail has hindered the development of a more detailed understanding of the unique biological properties associated with this material. The lack of any definitive structural information limits the development of this material as a useful pharmaceutical agent and thus increases the desirability of this information.